Chapter 5 96 Our finding of increased dynamin protein and Dnm1 and Dnm2 mRNA levels in proteinuric disease, combined with the results of Soda et al. showing that dynamin knock out results in proteinuria, leads to the following two hypotheses on dynamin’s role in GFB integrity. First, a minimum level of dynamin may be required for adequate GFB integrity; thus, if this level is not reached, GFB integrity is lost. Second, when the GFB is under stress, dynamin may be upregulated in an attempt to maintain or restore GFB integrity. We previously studied the expression of proteins required for proper GFB function in patients with acquired proteinuria and found changes that suggest a compensatory mechanism. (84) Thus, disease progression may occur when this compensatory mechanism becomes saturated or exhausted. In Dahl rats, which spontaneously develop proteinuria, we found that the levels of Dnm1 and Dnm2 mRNA are increased prior to the onset of proteinuria, a symptom that manifests only when protein is present in excreted urine. Proteinuria occurs when proteins pass through the GFB and are not sufficiently reabsorbed by the tubular system either due to saturation or malfunction of the tubular reabsorption process. Thus, the increase in glomerular Dnm1 and Dnm2 mRNA levels prior to the onset of proteinuria might reflect a compensatory mechanism in response to an early, presymptomatic increase in GFB permeability that does not yet lead to actual proteinuria. However, as proteinuric disease progresses, this compensatory system can become exhausted, thereby failing to prevent the onset of proteinuria. This hypothetical process is depicted schematically in Figure 4. Interestingly, we found that while animals that will become proteinuric (Dahl rats) express both more glomerular Dnm2 and Dnm1 mRNA before onset of proteinuria, this does not result in to an increase in dynamin protein. However, since dynamin is a regulatory GTPase, it is not necessary to have an increase in protein level to have an altered intracellular activity. Also, cathepsin L-mediated cleavage of dynamin may be increased and cause a decrease in dynamin protein staining.(13) In humans, we found that there is indeed an increase in glomerular cathepsin L protein which directly and strongly correlated with glomerular dynamin protein levels, as also observed by Sever et al.(13) Cleavage of dynamin by cytosolic L generates a 40 kDa N-terminal fragment of dynamin. As the used antibody recognizes an epitope of residues 822-838 of dynamin, these fragments were not identifiable in the immunohistochemistry experiments performed in this study. In our study, we found significantly higher levels of Ctsl mRNA in Dahl rats at all ages investigated, suggesting that in this rat model both the transcription and posttranslational cleavage of dynamin are increased.
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